Design, Synthesis, and Evaluation of 5-S-Aminoethyl-N6- azidobenzyl-5-thioadenosine Biotin Conjugate: A Bifunctional Photoaffinity Probe for the es Nucleoside Transporter

A bifunctional biotinylated photoaffinity label for the nitrobenzylmercaptopurine riboside (NBMPR)sensitive (es) nucleoside transporter (ENT1) has been synthesized and evaluated. This new probe, 5ʹ-Saminoethyladenosine-N6-azidobenzyl-5ʹ-thioadenosine biotin conjugate (SAEATA-14-biotin), exhibited high-affinity binding to the es transporter in K562 cells as determined by flow cytometry, with a Ki of 2.69 nM in competition against 5-(SAENTA)-x8-fluorescein. It also exhibited covalent linking to the es transporter in BeWo cell membranes upon UV irradiation. This new bifunctional probe is a potential tool for determining the amino acid residues involved in ligand binding at the NBMPR-binding site of the ENT1 nucleoside transporter, as well as for the purification of the transporter.